The invention concerns a process for the quantitative determination of illness specific antigens, in particular prostate specific antigens, as well as an apparatus for conducting this process.
Prostate specific antigen (PSA) is a tissue specific glycoprotein which can only be found in the prostate gland and its secretions. Because of the tissue specificity of PSA, it is well known to be a unique indicator for the prostate function as described by Hara M., and Kimura, H.: Two prostate-specific antigens, gamma-semino-protein and beta-microsemino-protein. J. Lab. Clin. Med. 1989; 113: 541-48; Graves, H. C. B., Kamarei, M., and Stamey T. A.: Identity of prostate specific antigen and the semen protein p30 purified by a rapid chromatography technique, J. Urol. 1990; 144:1510-5; and Sensabaugh, G. F., and Blake, E. T.: Seminal plasma protein p30: Simplified purification and evidence for identity with prostate specific antigen, J. Urol. 1990; 144: 1523-26. On the strength of these findings, PSA is therefore a particularly useful indicator in controlling patients after radical prostatectomy, and determining whether they suffer any further appearance of their prostate cancer. This because the operative intervention should have removed the entire prostate tissue and therewith also the possible source for releasing prostate specific antigen. In patients with a prostate cancer, in whom, through histological examination the cancer has been proven to be limited to the prostate organ, the value of PSA after radical prostatectomy falls under the detection level of the presently available immunoassays in 80-92% of the cases. A further increase in the PSA value is the earliest available indicator for a further appearance of the cancer after a complete prostatectomy. In this regard see Stamey, T. A., Yang, N., Hay, A. R., McNeal, J. E., Freiha, F. S., and Redwine, E.: Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N. Engl. J. Med. 1987; 317:909-16. Oesterling, J. E., Chan, D. W., Epstein, J. I., Kimball, A. W., Bruzek, D. J., Rock, R. C., Brendler, C. B., and Walsh, P. C.: Prostate specific antigen in the preoperative and postoperative evaluation of localized prostatic cancer treated with radical prostatectomy. J. Urol. 1988; 139:766-72. Oesterling, J. E.: Prostate specific antigen: A critical assessment of the most useful tumor marker for adenocarcinoma of the prostate, J. Urol. 1991; 145:907-23. Lightner, D. J., Lange, P. H., Reddy, P. K., and Moore L.: Prostate specific antigen and local recurrence after radical prostatectomy. J. Urol. 1990; 144:921- 26.
The presently available immunoassays for PSA can detect a PSA serum value of between 0.3 and 50 ng/ml. The sensitivity of the two leading commercially available immunoassays for PSA has a minimum analytical sensitivity of from 0.6 -0.8 ng/ml PSA. In this regard see Graves, H. C. B., Wehner, N., and Stamey T. A.: Comparison of a polyclonal and monoclonal immunoassay for PSA: Need for an international antigen standard, J. Urol. 1990; 144: 1516-22. In cases of a complete removal of a normal prostate, which became available by cystoprostatectomies due to causes other than prostate cancer, postoperative PSA values of less than 0.3 ng/ml have been found through the Yang Pros-check PSA. See Stamey, T. A., Kabalin, J. N., McNeal, J. E., Johnstone, I. M., Freiha, F., Redwine, E. A., and Yang, N.: Prostate specific antigen in the diagnosis and treatment of adenocarcinoma of the prostate, 11. Radical prostatectomy treated patients, J. Urol. 1989; 141:1076-83; Lange P. H., Ercole, C. J., Lightner, D. J., Fraley, E. E., and Vessella, R.: The value of serum prostate specific antigen determinations before and after radical prostatectomy, J. Urol. 1989; 141: 873-79; Hudson, M. A., Bahnson, R. R., and Catalona, W. J.: Clinical use of prostate specific antigen in patients with prostate cancer. J. Urol. 1989; 142: 111-17.
At this time an intensive effort is being made to find a sensitive immunoassay which detects a lower PSA level since such would be extraordinarily helpful for an early diagnosis of a recidivism of the prostate cancer in patients after radical prostatectomy. These ultrasensitive assays have a working area of from 0.1-1.2 ng/ml and thereby improve the analytical sensitivity by about three fold. Difficulties go along with such ultrasensitive assays. For example, the PSA calibrators in the lower regions (&lt;0.5 ng/ml) are not stabile if they are stored at about 4.degree. C., so that a calibration curve must be newly produced every day since a higher calibrator standard (for example an 8 ng/ml PSA calibrator) is weakened. The several day incubation times are expensive and demand strict temperature and bacteria contamination controls. See Graves, H. C. B., Wehner, N., and Stamey, T. A.: An ultrasensitive radioimmunoassay for prostate specific antigen. 1992, Clin. Chem.
The present invention therefore provides an improvement on the known type of processes and avoids the described disadvantages. It is generally useful with illness specific antigens; is technically simple in all laboratories; and in addition can be combined with other sensitivity improvement processes.
The process of the invention is principally based on a serum sample which is reduced through freeze drying to 1/5, 1/10 or 1/20 of its original volume. The volume of the extracted sample and the volume after reduction are determined. This factor is taken into consideration by the concentration calculation. The thusly reduced sample, which is considerably diminished in water content, but after as before, contains the prostate specific antigen in the same quantitative amount as the extracted sample, can then be analyzed in the customary way, and in particular, measured with traditional, commercially available immunoassays. Initial low concentrations henceforth appear in a measurable area of the immunoassay by the thusly obtained concentration. After the sample is measured with standard processes as before, the concentration of the sample from the extracted serum amount is determined by calculation.
To be sure, concentration through freeze drying is not fundamentally new, indeed, freeze drying is done regularly and frequently in order to raise the stability of proteins, tissues or other biological products, and in this manner makes the corresponding reagents durable and storable. In this way controls or reagents are treated in immunologically standard test processes. Another known and established process for freeze drying stems from the desire for weight or space savings. If sample water is removed, without changing its composition in other respects, it can be transported easier, and a complete, problem free reconstitution with water is possible. The principal of freeze drying can therefore also offer a considerable advantage from the viewpoint of storage (compare, for example U.S. Pat. No. 4,994,375). The stability of substances in long term storage can in general be maintained very well by freeze drying, and for the most part better, but at least as well as deep freeze storage, so that such a consideration of stability preservation likewise represents an important area of use for freeze drying.
New and surprising, is that despite the long and universal use of freeze drying measures, a process which uses such an effective means of concentration has not been used to attain an improved sensitivity in the measurement of, for example tumor indicators, such as illness specific antigens. Until now an improvement of the sensitivity of an analytical method predominantly would involve a change in the assay method. The reduction of samples, in order to thereby raise the concentration of the corresponding indicators, and to thereby convert the sample from background noise to the measurable area has heretofore not been done and also has not been reported in the literature.
Also of particular importance is an apparatus for conducting the process, which is characterized by a combination of several known devices. These include an apparatus for the determination of sample weight and/or volume, before and after freeze concentration; a calculator for the determination of the resulting concentration factors therefrom, as well as for back calculating the determined value of the extraction weight and volume of the sample from the patient; in conjunction with devices for conducting the freeze concentration itself and the final analysis of the immunological assays. Through a combination of these devices, a further automation of the described process for sensitivity improvement can be attained.
Therefore, according to the invention a process for the quantitative proof of prostate specific antigens by customary immunoassays is produced, which makes reliable analyses possible in low concentration areas. This process is characterized in that in the first instance a sample undergoes a freeze concentration and then thereafter an analysis or a standard assay is conducted, and that the therefrom resulting product is calculated back to its original sample volume.